PI(4,5)P₂-associated intoxication model: intracellularly-induced systemic death.

<p><b>A</b>) After translocation into the host cytoplasm by T3SS, ExoU (U) is postulated to bind to PI(4,5)P₂ residing on the inner leaflet of the plasma membrane. The PLA₂ activity of ExoU is activated by a cofactor (Co), ubiquitin or ubiquitinated proteins. <b>B</b>) ExoU hydrolyzes PI(4,5)P₂ and/or other phospholipid species (shown as Xs), leaving lyso-phospholipids, destabilizing the membrane. Reduction of PI(4,5)P₂ disrupts the anchoring and interaction between the focal adhesion, cytoskeletal structure, and plasma membrane, possibly through a cell signaling pathway. Cells round due to the weakened adhesion capability and actin filament depolymerization, leading to cytoskeletal collapse. The outer leaflet of the plasma membrane is intact at this stage. <b>C</b>) At a middle stage of infection, the plasma membrane starts to bleb, facilitating further membrane damage. <b>D</b>) During a late stage of infection, the outer leaflet of the plasma membrane is compromised, allowing the influx of an impermeable nucleic acid dye and staining of the nucleus (green), consequently large molecules (e.g. LDH) are released from lysed cells.</p

Intoxication of Host Cells by the T3SS Phospholipase ExoU: PI(4,5)P₂-Associated, Cytoskeletal Collapse and Late Phase Membrane Blebbing

<div><p><i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that is associated with hospital-acquired infections, ventilator-associated pneumonia, and morbidity of immunocompromised individuals. A subpopulation of <i>P. aeruginosa</i> encodes a protein, ExoU, which exhibits acute cytotoxicity. Toxicity is directly related to the phospholipase A₂ activity of the protein after injection into the host cytoplasm via a type III secretion system. ExoU enzymatic activity requires eukaryotic cofactors, ubiquitin or ubiquitin-modified proteins. When administered extracellularly, ExoU is unable to intoxicate epithelial cells in culture, even in the presence of the cofactor. Injection or transfection of ExoU is necessary to observe the acute cytotoxic response. Biochemical approaches indicate that ExoU possesses high affinity to a multifunctional phosphoinositide, phosphatidylinositol 4,5-bisphosphate or PI(4,5)P₂ and that it is capable of utilizing this phospholipid as a substrate. In eukaryotic cells, PI(4,5)P₂ is mainly located in the cytoplasmic side of the plasma membrane and anchors adaptor proteins that are involved in cytoskeletal structures, focal adhesions, and plasma membranes. Time-lapse fluorescent microscopy analyses of infected live cells demonstrate that ExoU intoxication correlates with intracellular damage in the early phases of infection, such as disruption of focal adhesions, cytoskeletal collapse, actin depolymerization, and cell rounding. At later time points, a membrane blebbing phenotype was prominent prior to the loss of the plasma membrane integrity and barrier function. Membrane blebbing appears to accelerate membrane rupture and the release of intracellular markers. Our data suggest that in eukaryotic host cells, intracellular ExoU targets and hydrolyzes PI(4,5)P₂ on the plasma membrane, causing a subsequent disruption of cellular structures and membrane integrity.</p></div

Enzymatic activity on PI(4,5)P₂ and high-affinity interaction of rExoU.

<p><b>A</b>) Enzymatic activity of rExoU on BODIPY-FL-PI(4,5)P₂ determined by TLC. The fluorescent substrate was incubated with 13.5 or 6.76 pmol rExoU in the presence of the cofactor ubiquitin (13.5 pmol), labeled as U + Ub. PI-PLC and honeybee PLA₂, 13.5 pmol each, were also tested. The position of the non-hydrolyzed substrate is indicated with an arrow. The percentage hydrolysis of each enzyme, averaged from 3 independent experiments, is shown below the chromatograph. <b>B</b>) Localization of ExoU-S142A in HeLa cells after infection in the presence or absence of the extracellular phospholipids. Infected cells were harvested, mechanically lysed, and fractionated. Fractions were analyzed by Western blot using an anti-ExoU monoclonal antibody. S: soluble fraction, M: membrane fraction. To achieve a linear detection range, 4-fold higher volumes of membrane fractions were loaded relative to soluble material. <b>C</b>) The Western blot signal intensity of both unmodified and ubiquitinated ExoU-S142A was quantified. Membrane fraction (dark gray) and soluble fraction (light gray) are shown as a 100% stacked column chart with the mean ± SD from 3 independent experiments. <b>D</b>) rExoU (13.5 pmol) binding to phospholipid (1600 pmol)-coated polystyrene plates detected by fluorescence microscopy. Magnification: 40x. <b>E</b>) rExoU affinity to phospholipids evaluated by an ELISA-based solid-phase binding assay. Polystyrene plates were coated with the indicated amounts of phospholipids. The binding of 13.5 pmol rExoU was determined using an anti-ExoU antibody and a horseradish peroxidase-conjugated secondary antibody. Peroxidase activity on the substrate ABTS with H₂O₂ was measured by absorbance at 405 nm. <b>F</b>) The Kd of rExoU to PI(4,5)P₂ determined with the ELISA-based binding assay. Nonlinear regression analysis of the concentration of rExoU (nM) as a function of total binding is shown. The plot represents the mean ± SEM of duplicates from 2 independent experiments. <b>G</b>) Binding of the PLCδ1-PH domain to PI(4,5)P₂ evaluated by nonlinear regression of the concentration of the PLCδ1-PH peptide (nM) as a function of total binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments. <b>H</b>) Inhibitory effect of rExoU to PLCδ1-PH binding to PI(4,5)P₂. rExoU (between 6.25 and 800 nM) was mixed into 50 nM of the PLCδ1-PH peptide for the solid-phase binding assay. IC₅₀: the concentration of rExoU to displace 50% of PLCδ1-PH binding. The plot represents the mean ± SEM of duplicates from 2 independent experiments.</p

Biological characteristics of ExoU-mediated toxicity to HeLa cells compared to other cell death mechanisms.

<p>*Cells were incubated with 1.35 to 675.65 pmol of rExoU in the presence of penta-ubiquitin (13.51 to 675.65 pmol) as the cofactor.</p>†<p>Cells were incubated with 67.57 pmol of rExoU in the presence of penta-ubiquitin (13.51 or 65.57 pmol) and 5 nmol PI(4,5)P₂.</p

<i>P. aeruginosa</i> PA103 strains used in this study.

<p><i>P. aeruginosa</i> PA103 strains used in this study.</p

Effect of PI(4,5)P₂ on ExoU-mediated cytotoxicity.

<p><b>A</b>) The addition of PI(4,5)P₂ increased ExoU-induced cell detachment from a culture plate. HeLa cells were pre-incubated with PI(4,5)P₂ or POPC or without additional phospholipids (no addition), infected with PA103ΔUT + <i>exoU</i> or <i>exoU-S142A</i>, and analyzed with a cell retention assay using crystal violet staining. <b>B</b>) Bacterial growth in the presence of additional phospholipids during infection. HeLa cells were preincubated with phospholipids for 1 h and infected. After 4 h, the culture medium was collected and subjected to a CFU assay. <b>C</b>) Increased efficacy of ExoU cytotoxicity in the presence of PI(4,5)P₂. HeLa cells were preincubated with the indicated amounts of phospholipids for 1 h, infected at MOI of 1.25 for 4 h to assess the early stage of intoxication. The influx of the impermeant propidium iodide represents cells with the compromised plasma membrane, which were quantified from micrographs of 3 independent experiments. <b>D</b>) PI(4,5)P₂ dose-dependency of ExoU cytotoxicity determined by the LDH release assay. HeLa cells were pre-incubated with indicated amounts of PI(4,5)P₂ for 1 h, infected at MOI of 2.5 for 4 h to capture the late stages of intoxication and cell lysis, and the release of LDH was measured in a 24-well format.</p

T3S-mediated translocation of ExoU-S142A and ExoU cytotoxicity in human epithelial cells.

<p><b>A</b>) After HeLa cell infection (at an MOI of 2.5 throughout Fig. 1), translocated ExoU-S142A was detected by Western blot analysis. The higher MW band is the ubiquitinated form of ExoU-S142A. uninf: uninfected, PA103ΔUT (ΔUT), PA103ΔUT + <i>exoU-S142A</i> (ΔUT + S142A), PA103ΔUT + <i>exoU-S142A</i> alone (bac). <b>B</b>) The percentages of ExoU-S142A-positive HeLa cells during infection quantified by flow cytometry. Cells were infected with PA103ΔUT + <i>exoU-S142A</i>, harvested at the indicated time points, and labeled with the anti-ExoU antibody U7.15 and Alexa Fluor 488-conjugated secondary antibody. <b>C</b>) LDH release from HeLa, A549, and fibroblast (Fb) cells representing the late stage of toxicity. Cells were infected with PA103ΔUT + <i>exoU</i> or <i>exoU-S142A</i> and released LDH was measured using a 6-well format at the indicated time points. <b>D</b>) The total percentage of intoxicated cells that includes detached cells from the culture plate and dead cells were quantified by flow cytometric analysis after staining with propidium iodide.</p

Phenotypes of cell death caused by apoptosis, surfactant application, and honeybee PLA₂.

<p><b>A</b>) Apoptotic, programmed cell death of HeLa cells induced by incubation with 3 µM gliotoxin. Apoptotic membrane blebbing was recorded by time-lapse microscopy for measurement analyses. Cell rounding (an arrow in the +4 min panel) and apoptotic blebbing (arrowheads in the +8 min panel) are highlighted. Cells were stained as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103127#pone-0103127-g002" target="_blank">Fig 2D</a>. Scale bar: 10 µm. Data shown are represenative of 3 experiments. <b>B</b>) Cell death caused by surfactant-based membrane rupture. To image the immediate action of the detergent, 100 µl of 0.1% Triton X-100 solution was gently dropped into the culture dish and the reaction of HeLa cells was followed by time-lapse imaging. Within 3 min, SYTOX green crossed the plasma membrane (an arrow in the +3 min panel). Scale bar: 10 µm. Data shown are represenative of 5 experiments. <b>C</b>) Intoxication of HeLa cells caused by the addition of 675 pmol honeybee venom PLA₂ to the dish. The plasma membrane was compromised within 5 min, as indicated by an arrow. Scale bar: 10 µm. Data shown are represenative of 5 experiments.</p

Disruption of focal adhesion as well as actin depolymerization, leading to cytoskeletal collapse.

<p><b>A</b>) The effect of ExoU on cytoskeletal structure and focal adhesion patterns in HeLa cells. After infection at MOI of 5 for 3 h 15 min, the actin cytoskeleton and a focal adhesion protein were visualized with phalloidin (green) and an anti-Zyxin antibody (red), respectively. Microtubules (MT) were immunolabeled with an anti-alpha-tubulin antibody (green). DAPI-stained nucleus is shown in blue. Scale bar: 10 µm. Data shown are represenative of 5 experiments. <b>B</b>) Depolymerization of actin cytoskeleton caused by ExoU. HeLa cells were infected with PA103ΔUT + <i>exoU</i> or <i>exoU-S142A</i> at MOI of 5 for 3 h 15 min, fixed, and stained with DNase I (G-actin, shown in green), phalloidin (F-actin, shown in red), and DAPI (blue). ExoU induced the bundled or collapsed morphology of actin filaments (arrows) and actin depolymerization, indicated as an intense G-actin staining pattern (arrowheads). Right bottom panels: Overlay images. Scale bar: 10 µm. Data shown are represenative of 3 experiments. <b>C</b>) Live imaging of ExoU-mediated depolymerization of the actin cytoskeleton prior to cell death. HeLa cells expressing GFP-tagged actin were infected and the change in the filamentous actin-GFP was detected by time-lapse microscopy. Cells were also labeled with the CellMask plasma membrane stain (red). The depolymerization of filamentous actin-GFP is highlighted with arrows in the +10 and +14 min panels. Depolymerized actin-GFP molecules were released from the cell upon cell lysis (compare the arrowhead in the 4 hpi panel to the one in the +8 min panel). Scale bar: 10 µm. Data shown are represenative of 10 experiments. <b>D</b>) The influence of ExoU on the focal adhesion protein Talin prior to plasma membrane damage. HeLa cells expressing a GFP-fused Talin signal peptide were infected, stained, and analyzed as described in Fig 6C. The GFP-Talin at the edge of cells began detaching from the culture dish and was pulled away from the plasma membrane (compare the arrows in the 4 hpi panel to the +13 min panel). Loss of cell adhesion led to cell rounding and then plasma membrane blebbing (arrowheads in the +26 and +39 min panels). Scale bar: 10 µm. Data shown are represenative of 4 experiments.</p

Five distinct mechanisms in which bacterial proteins manipulate the host ubiquitin system.

<p>Each piece of the pentagon illustrates a known mechanism for the intersection between pathogen effectors and the host ubiquitin system. Bacterial proteins utilizing each specific mechanism are listed. All bacterial effectors are depicted in yellow. Host target proteins are identified when known and labeled as (?) when unknown. E2, E2 conjugating enzyme, red; E3, E3 ligating enzyme; DUB, deubiquitylating activity, green; UB, monoubiquituin or ubiquitin chains, black ovals; NF-κB, nuclear factor kappa B; IκBα, I kappa B alpha; PLA₂, phospholipase activity with A₂ specificity.</p